Dissemination of carbapenemase-producing Enterobacterales in the community of Rawalpindi, Pakistan.

Carbapenems are considered last-line beta-lactams for the treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, their activity is compromised by the rising prevalence of carbapenemase-producing Enterobacterales (CPE), which are especially marked in the Indian subcontinent. In Pakistan, previous reports have warned about the possible spread of CPE in the community, but data are still partial. This study was carried out to analyse the prevalence of CPE, the genetic characterisation, and phylogenetic links among the spreading CPE in the community. In this cohort study, we collected 306 rectal swabs from patients visiting Benazir Bhutto hospital, Rawalpindi. CPEs were screened by using ertapenem-supplemented MacConkey agar. Identification was performed by using conventional biochemical tests, and genomes were sequenced using Illumina chemistry. Antibiotic resistance genes, plasmid incompatibility groups, and Escherichia coli phylogroups were determined in silico. Sequence types were determined by using MLST tool. The prevalence of CPE carriage observed was 14.4% (44/306 samples). The most common carbapenemase-encoding gene was bla-NDM-5 (n = 58) followed by blaNDM-1 (n = 7), blaNDM (non-assigned variant, n = 4), blaOXA-181 (n = 3), blaOXA-232 (n = 3) and blaNDM-7 (n = 1). Most of the CPE were E. coli (55/64, 86%), and the genomic analysis revealed a pauciclonal diffusion of E. coli with ST167 (n = 14), 405 (n = 10), 940 (n = 8), 648 (n = 6) and 617 (n = 5). We obtained a second sample from 94 patients during their hospital stay in whom carriage was negative at admission and found that 7 (7.4%) acquired a CPE. Our results indicate that the prevalence of CPE carriage in the Pakistani urban community was high and driven by the dissemination of some E. coli clones, with ST167 being the most frequent. The high CPE carriage in the community poses a serious public health threat and calls for implementation of adequate preventive measures.

Introduction

Enterobacterales reside as commensals in gut microbiota at concentrations of approximately 108 colony-forming units (CFU) per gram of faeces [1]. However, they may act as opportunistic pathogens frequently in community and hospital-acquired infections [2], causing meningitis, septicemia, pneumonia, pyelonephritis, peritonitis, and device-related infections [3, 4]. Some important virulence factors involved in pathogenicity of members of Enterobacterales are enterotoxins, hemolysins, toxins, fimbriae, mannose binding type1-H adhesion, intimin, alkaline phosphatase, haemagglutinin, siderophores lipopolysaccharide (LPS), capsular polysaccharide and biofilm production [5-7]. Virulence factors support bacteria to adhere, invade and damage host cells, escape host defense mechanisms and cause infection [8].

Resistance to antibiotics in Enterobacterales has been on the rise since the past two decades due to the large diffusion of CTX-M-type extended spectrum beta-lactamases (ESBL) which confer resistance to most β-lactams except for carbapenems [9]. Accordingly, the latter are commonly used as last line antibiotics for treating infections caused by multidrug resistant Enterobacterales including those producing ESBLs. Hence, the use of carbapenems has grown in parallel to the rise of ESBL-producing Enterobacterales, paving the emergence and dissemination of carbapenem resistance. While Enterobacterales can resist to carbapenems by combining the production of ESBL and decreased outer-membrane permeability, the main mechanism for carbapenem resistance is the production of carbapenemases [10, 11]. The major carbapenemases in Enterobacterales belong to three classes, Ambler class A (e.g., Klebsiella pneumoniae carbapenemase, KPC), class B (e.g. New Delhi metallo β-lactamase, NDM) and class D (e.g. OXA-48 and its close relatives such as OXA-181) [12]. Among them, NDM is one of the most commonly identified carbapenemase worldwide and many studies have reported its high prevalence in the Indian subcontinent [13-15]. Moreover, carbapenemase-encoding genes are carried on plasmids which often harbour multiple other antibiotic resistance genes (ARGs), narrowing the range of treatments possibly active on CPE [16].

E. coli and Klebsiella pneumoniae are the most frequent carriers of blaNDM, with specific sequence types (ST) such as ST11, 14, 15, 147 for K. pneumoniae and ST167, 410, 617 for E. coli. Diverse variants of carbapenemases such as VIM, IMP, NDM, KPC, and GIM have also been reported in Enterobacterales across Pakistan [17-19]. Limited data are available regarding the epidemiology of CPE carriage in the community [20-22], but suggesting a marked dissemination of CPE in Pakistan, Our study aimed to fill these gaps in assessing the CPE carriage prevalence in a Pakistani urban community and the underlying genetic determinants responsible for emergence of carbapenem resistance.

Methods

Ethics

The study was approved by the Ethical Review Committee, Rawalpindi Medical University on September 23, 2017. Verbal informed consent was obtained from all participants before the collection of samples, and it was written in participant’s record. Patients who refused to participate in the study were excluded. Participation was voluntary, and no minor was included in our study.

Patients and samples

Between January 2018 and May 2019, patients admitted to traumatology and gynaecology units of Benazir Bhutto Hospital, Rawalpindi, Pakistan with no antibiotic exposure, hospitalisation, and travel history abroad during the 3-months preceding the admission were included. Rectal swabs (Nuova, Canelli, Italy) were collected within 24 hours of admission. Patients in whom the first swab was negative for carbapenem-resistant Enterobacterales (CRE), a second swab was taken 72 hours after admission.

Bacterial isolation and identification

We initially did experiments to set the optimum culture conditions to recover CPE with respect to the epidemiological specificities of Pakistani CPE. We cultured already characterised, 4 strains of E. coli (2 each ertapenem-sensitive and ertapenem-resistant) [23] on MacConkey agar (Oxoid, Basingstoke, England) with increasing ertapenem (Merck & Co., Whitehouse Station, USA) concentrations ranging from 0.125 mg/L to 5 mg/L. We finally set the ertapenem concentration at 0.5 mg/L as the optimum concentration at which only resistant bacteria were growing while sensitive were inhibited.

Rectal swabs were thoroughly mixed with 1 mL normal saline and 100 μL of each sample was plated onto 0.5 mg/L ertapenem–supplemented MacConkey agar and incubated overnight at 37°C. Colonies with distinct morphotypes (with respect to the colour, size, and shape) were sub-cultured on MacConkey agar and initial identification was performed based on colony characteristics, Gram staining (Daejung Chemicals Ltd., Siheung, Korea), and conventional biochemical tests. All the Gram-negative short rods were further tested for Enterobacterales through a range of biochemical tests [24], using, oxidase (BDH Laboratory Supplies, Poole, UK), sulfide Indole Motility (SIM) medium (Liofilchem, Roseto, Italy), triple Sugar Iron (TSI) agar (Liofilchem, Roseto, Italy), methyl red-Voges-Proskauer (MR-VP) broth (Liofilchem, Roseto, Italy), nitrate broth (Liofilchem, Roseto, Italy), urea broth (Liofilchem, Roseto, Italy) and Simmon’s citrate media (Liofilchem, Roseto, Italy) [25]. After identification, pure bacterial colonies were stored in charcoal swabs (Nuova, Canelli, Italy) and shipped at ambient temperature to the bacteriology laboratory of the Bichat-Claude Bernard University Hospital, Paris, France. There, the bacterial species identification was confirmed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS, Bruker, Bremen, Germany).

Antimicrobial susceptibility testing

Enterobacterales isolates were tested for their susceptibility to 16 antibiotics from different antimicrobial groups: Penicillins: ticarcillin (75 μg), temocillin (30 μg), β lactams-β-lactamase inhibitor combination: amoxicillin-clavulanic acid (30 ug), ticarcillin-clavulanic acid (85 μg), Cephalosporins: cefoxitin (30 μg), ceftazidime (10 μg), cefotaxime (5 μg), cefepime (30 μg), Monobactams: aztreonam (30 μg), Carbapenems: ertapenem (10 μg), imipenem (10 μg), Aminoglycosides: amikacin (30 μg), gentamicin (10 μg), Quinolones: nalidixic acid (30 μg), ofloxacin (30 μg) and Sulfonamides: trimethoprim /sulfamethoxazole (30 μg) using the disk diffusion method (disks from I2A, Montpellier, France) on Mueller Hinton agar (Bio-Rad, Marne-la-Coquette, France) following the recommendations of the French committee for antimicrobial susceptibility testing based on the European Committee on Antimicrobial Susceptibility Testing (CASFM/EUCAST, May 2019) guidelines. Polymyxins: colistin MICs were measured by using the broth microdilution method (Biocentric, France). When Enterobacterales from a common sample shared the same antibiotic susceptibility pattern, they were assumed to be identical and only one was sent for sequencing.

Molecular methods

Genomic DNA of 78 CRE isolates based on resistance pattern of antimicrobial susceptibility profile was extracted using the EZ1 DNA Tissue Kit (Qiagen, Courtaboeuf, France). Libraries were prepared by using Nextera DNA Flex library preparation kit (Illumina, SanDiego, CA). The DNA was quantified using the Qubit Fluorometer (Thermo Fischer Scientific, Asnières sur Seine, France). Whole genome sequencing was performed on NextSeq 550 system (Illumina) with a mid-output kit (2x150 bases). Plasmids DNA of three different E. coli ST167 isolates were extracted with the Plasmid Midi Kit (Qiagen) for MinION system sequencing (Oxford Nanopore Technologies [ONT], Oxford, UK). Libraries were prepared with the Rapid Barcoding Kit, SQK-RBK004 (ONT) then plasmids DNA sequencing was performed on a Flongle Flow Cell (R9.4.1) on a MinION device (ONT).

Multilocus sequence typing (MLST)

MLST profiles were checked by using MLST tool (v2.16.4) [26, 27]. Data were compiled and evaluated, and isolates were given allele numbers, and sequence types (STs). For E. coli, it was done using the Warwick MLST database (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli) [28].

Bioinformatic analysis

Reads quality was assessed by FastQC v0.11.8 [29]. Trim Galore v0.4.5 was used for quality and adapter trimming (reads with Phred quality score inferior to 20 and length inferior to 50 bases were discarded) [30]. MetaPhlAn2 was used to confirm the phenotypic identifications of the strains and to detect putative cross-contaminations [31]. Reads were assembled using SPAdes v3.11.1 [32]. The quality of the assemblies was examined by using QUAST v5.0.2 [33]. ARGs and plasmids replicons were searched by using Abricate v0.9.8 using AMRFinder database (2019-04-29) and PlasmidFinder database (2019-08-28), respectively, with 80% minimal coverage and nucleotidic identity filters [34, 35]. Chromosomal point mutations conferring quinolones resistance in E. coli and K. pneumoniae were searched with Resfinder 4.1 on the Center for Genomic Epidemiology website (http://www.genomicepidemiology.org)Core genomes were determined by multi-alignment tool Parsnp v1.2 [36]. The single nucleotide polymorphisms (SNPs) based phylogenetic tree was constructed with PhyML v3.0 using General Time Reversible (GTR) model and 1000 boostraps [37]. Plasmids were reconstructed by hybrid assembly of Nanopore and Illumina reads with Unicycler v0.4.9b [38]. ARGs and plasmids replicons were searched by Diamond v0.9.22.123 [39] with AMRFinder database (2019-04-29) and CD-HIT v4.7 [40] with PlasmidFinder database (2019-08-28), respectively. The Illumina reads of each isolate were mapped onto the reconstructed plasmid genomes with BWA v0.7.17 [41]. A plasmid was considered to be present in a genome if the Illumina reads covered >94% of the plasmid sequence with 10X depth and with maximal mapping quality. The 94% threshold was set according to the lowest coverage observed for the mapping of Illumina reads against the plasmid sequence within the same strain.

Quantification of CRE

Rectal samples thoroughly mixed with 1 mL normal saline were serially diluted till 10−6 and plated onto MacConkey agar with 0.5 μg/mL ertapenem and without ertapenem and incubated overnight. Colony-forming units (CFU) were counted in decimal logarithms at the dilution in which 1 to 100 CFU were grown. The CRE relative abundance (CRE-RA) was calculated as the ratio of the CRE counts divided by the total number of Gram-negative bacteria, expressed as a percentage. For patients who were found to carry more than one CRE clone, the CRE-RA of the dominant clone was considered.

Statistical analysis

Statistical analysis of relative abundance was performed using Wilcoxon and Kruskal-Wallis tests [42]. Differences of CRE-RA between community-acquired CRE and hospital-associated CRE were calculated by using Wilcoxon test, while Kruskal-Wallis test was used to calculate differences between community-acquired bacterial species and between the different community-acquired E. coli sequence types [43].

Results

Sampling

A total of 306 rectal swabs (one per patient) were collected within 24 hours of their admission to the traumatology and gynaecology wards of Benazir Bhutto hospital. Among them, 27.8% (n = 85) samples were from male patients from the traumatology ward, 21.9% (n = 67) from female patients from the traumatology ward and 50.3% (n = 154) were from female patients from the gynaecology ward. The average age for male and female trauma patients was 38 years and 44 years respectively, while for female gynaecology patients the average age was 28 years. From 94 patients who were subsequently hospitalised and in whom the first swab was negative on the ertapenem-supplemented MacConkey agar, a second swab was taken after 72 hours of their hospital stay (Fig 1). The mean duration between admission and follow-up sampling was eight days (range 3–60 days). Most of the swabs were collected during three periods of time: January to May 2018 (70/306, 23%), August to November 2018 (104/306, 34%), January to May 2019 (132/306, 43%) (Fig 2).

Fig 1

Flowchart of the study.

Fig 2

Distribution of CPE positive and negative samples by month of collection, (January 2018 to May 2019).

Phenotypic characteristics of the recovered isolates

Enterobacterales isolates were Gram-negative, pink colored, short rods under the microscope. On MacConkey agar E. coli colonies appeared as pink, flat, dry and non-mucoid surrounded with darker pink zone of precipitated bile salts, K. pneumoniae were mucoid, and pink. C. freundii, and E. cloacae also formed pink colonies due to lactose fermentation. Biochemical tests results were interpreted according to Clinical Microbiology procedures handbook [24].

Prevalence of CPE carriage

A total of 170 Enterobacterales isolates was cultured on ertapenem-supplemented MacConkey agar, among which 78 were left after deduplication (based on their antibiotic susceptibility profile). After genomic analysis, 73 were found to produce at least one carbapenemase-encoding gene. Among the 306 patients screened at admission, 48 were community acquired CRE, and 44 carried a CPE, yielding a CPE carriage rate of 14.4% (44/306) in the community (Fig 1). The carriage rate was 16.5% (14/85) among traumatology-admitted male patients, 19.4% (13/67) among traumatology-admitted female patients and 11.0% (17/154) among gynaecology-admitted female patients. All samples were from patients who were not showing any infectious disease symptoms and they were not receiving any medication at the time of sampling.

All the community-carried (n = 69) and hospital-acquired CPE isolates (n = 9) were found to be resistant to ceftazidime, cefotaxime, ertapenem, and cefoxitin while 1 strain (231A) remained apparently intermediate to cefepime. Imipenem was inactive against 92% strains while 79% isolates were resistant against aztreonam. For quinolones, 99% resistance was found against nalidixic acid and 97% against ofloxacin. Trimethoprim/sulfamethoxazole was found resistant against 94% strains, while a lower rate of 24% and 31% resistance was observed for amikacin and gentamicin, respectively. Only 1 isolate (201A) found resistant to colistin (S1 Table), MIC measured for this isolate was 4 mg/L and it was found phenotypically resistant to all antibiotics used except imipenem and amikacin. Bacterial isolates were grouped into extensively-drug resistant (XDR) and multi-drug resistant (MDR) based on the resistance phenotypes definitions of Magiorakos et al. [44]. According to these definitions, none of the isolates was PDR, while 64 (82%) isolates were grouped into XDR and 14 (18%) isolates into MDR category (S2 Table).

Antimicrobial resistance genes

A total of 78 CRE isolates were subjected to whole genome sequencing, among which 73 were carbapenemase producing Enterobacterales (CPE) while 5 strains did not carry any carbapenemase gene while phenotypically all isolates were resistant to carbapenems. Among the 73 Enterobacterales strains, 56 distinct antibiotic resistance genes were identified, including 17 encoding β-lactamases (Fig 3). The most common carbapenemase gene was blaNDM-5 (n = 58) followed by blaNDM-1 (n = 7), blaNDM (unidentified variants, n = 4), blaOXA-181 (n = 3), blaOXA-232 (n = 3) and blaNDM-7 (n = 1). The three strains carrying blaOXA-181 were Citrobacter freundii which also carried blaNDM-1. The blaNDM-5 gene was only found in E. coli while blaOXA-232 was only found in K. pneumoniae. Among the hospital-associated strains (n = 9), eight carried blaNDM-5 and one carried blaNDM-1. Forty-three strains were found to harbour an ESBL-encoding gene, mainly CTX-M-15 ESBLs (one exception being CTX-M-139, which is a variant of CTX-M-15) (Fig 3 and S3 Table). The main plasmid-mediated quinolone resistance (PMQR) genes were qnrS1 (n = 12) and qepA (n = 3) (Fig 4). Consistent with their resistance phenotype, 66/68 E. coli had mutations in topoisomerases known to confer resistance to quinolones. On the other hand, no mutation was found in 2/68 quinolone-resistant E. coli and 3/3 quinolone-resistant K. pneumoniae. These five isolates carried PMQR genes (Fig 4). Multiple aminoglycoside resistance genes (ARG) coding for enzymes that inactivate the antibiotic were identified in 72/73 strains: aadA2 (n = 46), aadA1 (n = 19), aadA5 (n = 12), aph(3’’)-Ib (n = 12), aph(6)-Id (n = 12), aadA16 (n = 7), and aac(3)-IIa (n = 4) (Fig 5). Phenotypic resistance rates were low for amikacin and gentamicin since the antibiotic spectra of these ARGs are different. 16S rRNA methyltransferase genes, which confer resistance to all aminoglycosides, were found in 18 strains: rmtB1 (n = 12), rmtF1 (n = 3), armA (n = 3). The sul (dihydropteroate synthase) and dfr (dihydrofolate reductase) resistance genes that confer cotrimoxazole resistance were present in 67 and 73 strains, respectively, consistent with phenotypic resistance. Only one isolate (201A) carried the colistin resistance gene mcr-1 (Fig 6). Same isolate was also found resistant to colistin in vitro. The correlation between the observed phenotypic antibiotic resistance pattern and genomic content of antibiotic resistance genes (ARGs) is presented in supporting information (S4 Table).

Fig 3

Acquired beta-lactamase–encoding genes.

Carbapenemase-encoding genes are highlighted in red.

Fig 4

Bar plot depicting the distribution of plasmid-mediated quinolone resistance genes.

Fig 5

Bar plot depicting the distribution of aminoglycoside resistance–encoding genes.

Fig 6

Phylogeny, STs, resistance genes and plasmids of E. coli.

*: hospital-acquired isolates.

MLST of community-acquired E. coli identified 18 different STs (S3 Table, Figs 6 and 7). The most prevalent were ST167 (phylogroup A, 14/60), ST405 (phylogroup D, 10/60), ST940 (phylogroup B1, 8/60), ST648 (phylogroup F, 6/60) and ST617 (phylogroup A, 5/60) (S3 Table). Hospital-associated E. coli (n = 8) were divided into five different STs except for two ST167 strains and two ST2659 strains (Fig 6). The three K. pneumoniae in the study were isolated from three different community subjects and all belonged to ST231. The three Enterobacter cloacae in the study (two community-acquired, one hospital-associated) belonged to ST93. The four C. freundii were of community origin and belonged to ST396 (n = 1) and ST107 (n = 3).

Fig 7

Pie chart of the sequence types (ST).

The distribution of STs among carbapenemase-producing E. coli from the community.

Phylogeny

The core genome of the 68 E. coli was made of 2,094 genes (1,807,969 bases in total). The five most frequent STs of community-acquired E. coli made distinct clades (Fig 8). The maximal genetic distances between isolates of the same clade were 1,954 SNPs for ST167, 1,606 SNPs for ST405, 3,098 SNPS for ST940, 3,101 SNPs for ST617 and 3,679 SNPs for ST648. Among all the E. coli, the most genetically distant were separated by 37,852 SNPs. Phylogenetic analysis revealed that community and hospital acquired were closely related isolates suggesting that the community reservoir of CPE is fueling the hospital reservoir. Among ST940 and ST167 community and hospital acquired isolates were clonal sharing less than 10 SNPs.

Fig 8

Phylogenetic tree of E. coli.

*: hospital-associated isolates. The dendrogram showing the phylogenetic relationship between community-acquired and hospital-associated E. coli.

Replicon typing and plasmid analysis

We identified 27 plasmid replicons in the 78 genomes. The IncX3 and IncFII incompatibility groups commonly associated with blaNDM were present in 23% (18/78) and 41% (32/78) strains, respectively (Fig 9). To assess the diversity of plasmids within the ST167 strains, we sequenced and reconstructed plasmids from three different ST167 E. coli isolates (46D, 60E, and 279D) that had different genetic backgrounds based on the resistance genes, phylogenetics and plasmids typing results. They were found to carry blaNDM on three different IncF plasmids and isolate 46D possessed blaNDM-encoding IncF plasmid with another blaNDM-encoding IncN replicon plasmid. Then, we mapped the Illumina reads of all the E. coli ST167 on the reconstructed plasmids sequences. The four reconstructed plasmids were also found in the genomes of the other E. coli ST167 (Fig 10).

Fig 9

Distribution of plasmids replicons.

Fig 10

Screening of the four reconstructed plasmids within E. coli ST167 isolates.

Relative abundance

The mean relative abundance for CRE was 0.5% (median 0.1%, min 0.0003% and max 3.4%). There were no significant differences of CRE-RA between community-acquired CRE and hospital-associated CRE (Wilcoxon test, p = 0.2) (Fig 11), between community-acquired bacterial species (Kruskal-Wallis test, p = 0.29) (Fig 12) and between the different community-acquired E. coli sequence types (Kruskal-Wallis test, p = 0.92) (Fig 13).

Fig 11

CRE relative abundance of community-acquired and hospital-acquired CRE.

Fig 12

CRE relative abundance of the different species of Enterobacterales.

Fig 13

CRE relative abundance of E. coli ST167 versus other ST of E. coli.

Discussion

The main result of this study is the high prevalence of CPE carriage (14.4%, 44/306) among patients attending the traumatology and gynaecology wards of the Benazir Bhutto hospital of Rawalpindi. This high prevalence rate is in line with previous reports [20, 21, 45]. High community carriage can lead towards longer hospitalization, higher medical cost, and increased mortality due to limited treatment options. Our study results suggest that community carriage is also one of the important factors for CPE dissemination in hospital settings. A second sample was taken from 94 patients (negative at admission) during their hospital stay and it was found that 7 (7.4%) acquired a CPE. Phylogenetic analysis revealed that there is genetic relatedness of hospital acquired isolates with few community acquired isolates suggesting a person to person spread or same source of contamination. Same clones were circulating in community and hospital settings. In hospital environment, antimicrobial exposure, longer hospitalization, the use of invasive devices, and severe underlying infections are considered independent risk factors for carbapenem producing Enterobacterales, but our study shows increasing CPE carriage among community along with the detailed genomic analysis to understand the determinants responsible for the spread of CPE specially in community settings. Different studies including a review of 10 studies of different regions (Asia, Europe, and North America) reported prevalence of CRE ranging from 0.4% to 29.5% isolated from various samples including faecal samples from healthy individuals and outpatients showing that community-based CPE are increasing in Asia, particularly in Indian subcontinent [46-48]. In 2013, Nair et al, from India reported 12.3% prevalence of carbapenem resistance among Enterobacterale isolates from patients admitted to different wards, ICU, and visiting outpatient department in respective hospital [49]. Two main factors underlying the specific situation of CPE in Pakistan may be proposed. The first one is the uncontrolled use of antibiotics, several antibiotics can be purchased over-the-counter without prescription, thereby promoting self-medication and potential overuse, causing a big challenge to community health [50]. The second factor is that Pakistan is a developing country where access to clean drinking water and safe food is not secured for large part of population, which may facilitate the circulation of intestinal bacteria such as CPE. A study conducted in Pakistan on environmental water collected from different sites reported the presence of NDM-1 producing bacteria in water sources [51]. In India, the blaNDM was similarly found in tap water [52].

The diffusion of NDM in the community of Rawalpindi was driven by a limited number of E. coli STs, with 43/55 (78%) of NDM-producing E. coli belonging to the ST167, ST405, ST940, ST648, and ST617. The most frequent ST was ST167 which belongs to the phylogroup A. ST167 NDM-producing E. coli had already been spotted [53-55], yet the extent of its diffusion in this region had not been assessed to date. An Indian study conducted in 2020, on sequence types of cefotaxime resistant E. coli showing that ST167 was more prevalent in the community followed by ST410 and ST648 [56]. Another study conducted in sewage from Islamabad showed that ST648 was among prevalent STs in E. coli isolates [22]. A Chinese study analysed all bacterial genomes in the GenBank database containing blaNDM [13]. Among these genomes, E. coli was the most common species (n = 305) and the ST167 was the main ST (44/305) among those possessing blaNDM. In addition, E. coli strains of ST167 carrying blaNDM have been reported from China (hospital-outbreak), India, South Korea, Switzerland, South Africa, and the United States [13, 57]. ST167 carrying blaNDM-5 has been the most prevalent sequence type reported from China in clinical settings and has potential to become the main ST type worldwide [58, 59].

Combination of resistance genes, and ability to easily disseminate put E. coli ST167 as a potential "high-risk clone" [60] such as the CTX-M-15-producing E. coli ST131. Moreover, using nanopore sequencing technology, we were able to retrieve circularised plasmids for E. coli ST167 strains and among these isolates, we observed several blaNDM-encoding plasmids. The diversity of resistance plasmids observed within ST167 isolates indicates that carbapenem resistance is not spreading through a specific plasmid and that such as E. coli ST131, there does not seem to be a bottleneck for the blaNDM–bearing plasmids for the E. coli ST167.

The genomic analysis showed that blaNDM (90%) was the most prevalent carbapenemase gene in our study and blaNDM-5 was the most common variant (58/70, 82%). Previously, a study from Lahore (Pakistan) showed the presence of blaNDM-5 in isolates from neonates [61]. However, majority of studies showed that blaNDM-1 was more prevalent in Asia especially Indian subcontinent [13, 51, 57]. BlaNDM-5 seems to be becoming a prevalent NDM variant in the Indian subcontinent [14]. Apart from NDMs, the OXA-181 carbapenemase was found in three community acquired C. freundii belonging to ST107 from three different patients. These three strains were co-harbouring blaNDM-1 and blaOXA-181 and their resistance genes and plasmid content were also similar. Previous studies in Pakistan have reported the presence of the mcr-1 gene in migratory birds, clinical settings, and healthy broilers [62-64]. We report here the first detection of mcr-1 from an outpatient in Pakistan. In countries like Pakistan, where the prevalence of CPE is high, increasing resistance against last-line antibiotics such as colistin is of major concern. Spread of mcr genes in CRE is a potential concern as it could create CRE strains that would be potentially pan-drug resistant. Although only one CRE isolate in this study was found to be carrying colistin resistance gene, it signifies that surveillance and monitoring should be conducted on regular basis to slow down the emergence of such strains. A study from Thailand also reported similar findings of mcr-1 detection from clinically isolated CRE during 2016–2019 [65]. Dissemination of mcr-1 together with blaNDM in Asian counties is alarming and it needs attention.

The average age for male and female trauma patients was 38 years and 44 years respectively, while for female gynaecology patients the average age was 28 years. According to a study age >50 years is one of the risk factors for carbapenem resistance [66]. Another study has reported high association of carbapenem resistance acquisition in children with age less than ten years [67]. Hence, we assessed the prevalence of CPE in a group with no specific risk factor for multidrug resistance carriage. There is possibility that the CPE prevalence could even be higher in specific populations such as young children or elderly people.

The mean relative abundance of CRE (CRE-RA) was similar to that found in other studies in Europe for community ESBL-producing E. coli [68, 69] highlighting that in patients with no recent exposure to antibiotics, multidrug-resistant Enterobacterales remain subdominant. Surprisingly though, the mean CRE-RA of hospital-acquired CRE was not higher. Still, we did not record the antibiotic intake after admission in these patients.

This study will be helpful in raising awareness among medical professionals, scientific community, and policy makers about recent antimicrobial resistance trend in community and need for solutions by limiting the use of antimicrobials and switching towards alternative options such as, vaccines, immunotherapeutics, and modulation of gut microbiota [70].

This work has certain limitations. The population considered might not be a true reflection of community, even though we only included patients who reported no antibiotic intake nor hospitalisation in the 3-months preceding the visit. Also, we used rectal swabs instead of faeces for feasibility matters which may have resulted in underestimation of CPE carriage. Accordingly, we may have overestimated the hospital acquisition rate in that some patients deemed negative for CPE at admission may have been false negative. Screening on ertapenem supplemented MacConkey agar may have led to underestimation of total Enterobacterales as a substantial number of phenotypically susceptible (may carry genotypic resistance markers) strains may have been excluded.

Conclusion

We observed a high carriage rate of CPE among community patients in Rawalpindi, mostly driven by a limited number of E. coli STs among where ST167 was dominant. Asymptomatic CPE carriers are the potential source of its dissemination in community, hospitals as well as in environment as phylogenetic analysis indicated that same CPE clones were circulating in community and hospital settings. Successful dissemination of Enterobacterales clones in community, continuous transmission of carbapenemase harboring plasmids through mechanism of horizontal gene transfer, and co-occurrence of multi-drug resistance genes, are the main reasons for increased carbapenem resistance in community. Stopping dissemination is important otherwise, CPE will achieve a foothold in community and hospitals. CPE transmission can be controlled by active surveillance, raising awareness, regular screening, and implementation of infection control strategies.

Antimicrobial susceptibility testing results.

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Categorization of CRE isolates into XDR and MDR.

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Acquired antibiotic resistance genes, sequence types, species, and phylogroups.

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Correlation between the genomic content of antibiotic resistance genes (ARG) and observed phenotypic resistance.

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6 Dec 2021

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Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

Reviewer #3: N/A

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

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PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

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Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I have some question/comments for the author to improve their manuscript.

1. Introduction: Situation of CPE/CRE in Pakistan should be mentioned.

2. Line 110-118: How many isolates were selected? and what is the criteria to select?

3. Line 178-185: What is colistin susceptibility result? it is not mention in this part.

4. Line 230: % should be shown.

5. Discussion: Although this study showed only one case/isolate of mcr-harboring CPE but it is a signal to monitoring. The mcr-harboring CRE were documented in several literature. The author can discuss this detail to lead awareness of the clinician and infection control. Please see 'Paveenkittiporn et al., Whole-Genome Sequencing of Clinically Isolated Carbapenem-Resistant Enterobacterales Harboring mcr Genes in Thailand, 2016-2019. Front Microbiol. 2021 Jan 11;11:586368. doi: 10.3389/fmicb.2020.586368.". This might be useful to discuss because it report from the Asian country.

Reviewer #2: Reporting data on the detection of CPE in each country is necessary to understand the dissemination of CPE and to develop countermeasures. However, the description of the results is not clear in the text. It makes difficult to understand results and needs to be revised.

L. 186 - 208

The number of CPE is unclear. How many CPE strains were isolated, 73, or 78? Which genes were detected in the strains? Which species were detected? Authors should add a table summarizing the essential information from Fig. S3 to the text.

Methods, Patients and samples

Although the paper is about CPE in the Community, it includes strains detected after 72 hours of hospitalization. These should be excluded.

L. 142 - 150

There is a problem with CRE quantification because the counts are made using cultured bacteria that have been previously subjected to ertapenem selection, which overestimates the number of CRE. Also, CRE quantification is not used for discussion and I do not understand the significance of its inclusion. It would be better to delete the descriptions and figures related to CRE quantification.

L. 215

Strains named Enterobacter cloacae complex should be re-identified using ANI and the correct species name filled in. Also, do not abbreviate the name of a genus that appears for the first time.

Fig.1

In the process of processing the 262 negative patients, 4 patients (negative:262-refused:98-not-hospitalized:63-ICU:3-sampled:94) are missing. Please check.

Fig.3

It should be carefully explained where the "clade" is. Especially for the ST648 clade, it is difficult to understand where the clade is located on the tree. It would be better to add some outgroups and make root for easier reading.

In the stacked bar graph of Fig. S1, the number of samples and the results are stacked on top of each other, and the number may be twice as large. It needs to be redesigned.

In Fig. S2, S4, and S5, only the number of detections is listed, and information such as from which ST type of which bacteria species is not available. Please add this information.

Reviewer #3: In this study the authors investigated the prevalence of carbapenemase-producing Enterobacterales (CPE) in patients attending a hospital from the community in Rawalpindi, Pakistan. Rectal swabs obtained from patients attending traumatology and gynaecology services without any preceding antibiotic exposure, hospitalisation or travel history in the last 3 months were plated on screening agar. Enterobacterales were identified by MALDI-TOF MS and antibiotic susceptibility testing and whole-genome sequencing performed.

The paper is well written and all the results are clearly presented. It presents valuable information on the prevalence of community CPE in Pakistan. WGS highlights the dominant sequence types carrying CPE genes and provides epidemiological information on plasmids carrying carbapenemase genes.

I have the following comments and suggestions for the authors:

METHODS:

Page 5, line 98: Antimicrobial susceptibility testing. It would add to the manuscript if the authors could perform susceptibility testing against newer agents, like aztreonam+avibactam and cefiderocol.

RESULTS:

Sampling: Page 8, line 160-162: In this study a young patient cohort was investigated (average age 38 and 44 years). How does this compare to other studies from the region? Could this influence the finding?

Prevalence of CPE carriage: Page 8, line 169: It would be interesting to add some information on whether the high community prevalence was reflected in disease burden in the hospital. How many of the patients required treatment for CPE? Were any of the hospital acquired cases considered to be linked?

Phylogeny: Page 10, line 220: The authors comment on the maximal genetic distances but do not discuss genetic similarities. On the tree some of the sequences look like they are related? Did they find possible relatedness between community and hospital acquired strains indicating possible cross transmission? Could the authors comment on this?

Relative abundance: Page 11, line 239: The authors present data on quantification of CRE but do not discuss the finding. Did they expect a difference in the relative abundance between community-acquired CRE and hospital-associated CRE? Does this depend on whether the patient receives antibiotics? I suggest to briefly mention this in the discussion.

DISCUSSION:

Line 248: ‘CPE carriage is mostly associated with hospital environment’ Did the other studies undertaken in Pakistan focus on hospital acquired cases? It would be interesting to discuss isolates that were acquired in hospital here. On the tree they look spread out over the clades but closely related to some of the community isolates? What is known about hospital acquisition of CPE in hospitals in Pakistan? How many of the CPE positive patients did require CPE treatment? What impact does the high community carriage rate have on hospitals?

Line 278: ‘Combination of resistance genes, virulence, and ability to easily disseminate…’ The authors mention virulence here but do not discuss virulence data for the observed ST types. How frequent is ST 167 found in clinical samples?

Line 295/296: It would add to the value of the manuscript to be able to discuss cefiderocol susceptibilities here. It might not be available as an agent in Pakistan yet but with the high prevalence of metallo-b-lactamases it would be helpful to assess its activity against the isolates of the study.

**********

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Reviewer #2: No

Reviewer #3: Yes: Kirsten Schaffer

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21 Jan 2022

RE: PLOS One

Title: Dissemination of Carbapenemase-producing Enterobacterales in the community of Rawalpindi, Pakistan

============================================================

Reviewer's comments:

Reviewer #1: I have some question/comments for the author to improve their manuscript.

1. Introduction: Situation of CPE/CRE in Pakistan should be mentioned.

Response: In revised version of manuscript, the information regarding CPE situation in Pakistan has been added. Please see lines 73 – 75 at page No. 4.

2. Line 110-118: How many isolates were selected? and what is the criteria to select?

Response: No. of isolates and selection criteria have been mentioned at page No. 5 (lines 113 -114).

3. Line 178-185: What is colistin susceptibility result? it is not mention in this part.

Response: Colistin susceptibility results have been added on Page No. 9 (lines189-191).

4. Line 230: % should be shown.

Response: The percentages of IncX3 and IncFII incompatibility groups commonly associated with blaNDM are mentioned in revised version on Page 11 (line 242).

5. Discussion: Although this study showed only one case/isolate of mcr-harboring CPE but it is a signal to monitoring. The mcr-harboring CRE were documented in several literature. The author can discuss this detail to lead awareness of the clinician and infection control. Please see 'Paveenkittiporn et al., Whole-Genome Sequencing of Clinically Isolated Carbapenem-Resistant Enterobacterales Harboring mcr Genes in Thailand, 2016-2019. Front Microbiol. 2021 Jan 11;11:586368. doi: 10.3389/fmicb.2020.586368.". This might be useful to discuss because it report from the Asian country.

Response: We have added details as suggested and thanks to the reviewer for suggesting this article. In the revised version, we have cited this paper in the discussion, to highlight the spread of mcr harboring CRE in Asian countries. (Page 15, lines 319-325).

-----------------------------------------------------------------------------------------

Reviewer #2:

Reporting data on the detection of CPE in each country is necessary to understand the dissemination of CPE and to develop countermeasures. However, the description of the results is not clear in the text. It makes difficult to understand results and needs to be revised.

Response: Your comments have been considered and revisions have been made throughout the text to have a clearer reading of the results.

L. 186 - 208

The number of CPE is unclear. How many CPE strains were isolated, 73, or 78? Which genes were detected in the strains? Which species were detected? Authors should add a table summarizing the essential information from Fig. S3 to the text.

Response: We have clearly mentioned the number of CPE in revised version. Page No. 9, lines 193-195. Fig. S3 has been converted to table (S1 Table) containing all essential information such as genes detected, species, sequence types and phylogroups.

Methods, Patients and samples

Although the paper is about CPE in the Community, it includes strains detected after 72 hours of hospitalization. These should be excluded.

Response: Yes, the paper mainly discusses the community carriage of CPE but we also followed up (where possible) for the acquisition of CPE during hospitalization. We consider that this data is also of value as it shows the genetic features of hospital associated isolates which were quite like community acquired isolates. Also, the reviewer # 3 has suggested to include the discussion of hospital acquired isolates, hence we are keeping this information in the revised version.

L. 142 - 150

There is a problem with CRE quantification because the counts are made using cultured bacteria that have been previously subjected to ertapenem selection, which overestimates the number of CRE.

Response: The samples have been cultured on ertapenem-supplemented media and have not been exposed to ertapenem prior to spreading onto the plate.

Also, CRE quantification is not used for discussion and I do not understand the significance of its inclusion. It would be better to delete the descriptions and figures related to CRE quantification.

CRE quantification has been included in Discussion. Page 15-16, L:333-337

L. 215

Strains named Enterobacter cloacae complex should be re-identified using ANI and the correct species name filled in. Also, do not abbreviate the name of a genus that appears for the first time.

Response: ANI of the three Enterobacter strains was calculated with FastAni (Jain, C., Rodriguez-R, L.M., Phillippy, A.M., Konstantinidis, K.T. Aluru, S. Nat Commun 9, 5114 (2018), and following were the results:

ANI of 99.9369 for 240Ea with Enterobacter cloacae,

ANI of 99.9451 for 255A with Enterobacter cloacae

ANI of 99.934 for 259B with Enterobacter cloacae.

Hence the three strains are E. cloacae and changes have been made accordingly in the revised manuscript. (Page No. 3, line 71), (Page No. 9, line 199) and (Page No. 11, line 224).

Fig.1

In the process of processing the 262 negative patients, 4 patients (negative:262-refused:98-not-hospitalized:63-ICU:3-sampled:94) are missing. Please check.

Response: These 4 patients were found carbapenem resistant on ertapenem supplemented agar but were not carrying any carbapenemase gene (CPE negative 262: refused:98-not hospitalized: 63-ICU:3-sampled:94,while remaining 4 were CRE not CPE, hence missing). We have made changes in Fig. 1 to clear this confusion.

Fig.3

It should be carefully explained where the "clade" is. Especially for the ST648 clade, it is difficult to understand where the clade is located on the tree. It would be better to add some outgroups and make root for easier reading.

Response: Changes have been made as suggested in Fig. 3.

In the stacked bar graph of Fig. S1, the number of samples and the results are stacked on top of each other, and the number may be twice as large. It needs to be redesigned.

Response: Fig. S1 has been redesigned. Number of samples and results have been separated.

In Fig. S2, S4, and S5, only the number of detections is listed, and information such as from which ST type of which bacteria species is not available. Please add this information.

Response: All the information of the isolates (ST types, bacterial species) has been provided in detail in Table S1,while the ST types and species have also been updated in Figures S2, S3 and S4 as per reviewer’s suggestion.

--------------------------------------------------------------------------------------------

Reviewer #3:

In this study the authors investigated the prevalence of carbapenemase-producing Enterobacterales (CPE) in patients attending a hospital from the community in Rawalpindi, Pakistan. Rectal swabs obtained from patients attending traumatology and gynaecology services without any preceding antibiotic exposure, hospitalisation or travel history in the last 3 months were plated on screening agar. Enterobacterales were identified by MALDI-TOF MS and antibiotic susceptibility testing and whole-genome sequencing performed.

The paper is well written and all the results are clearly presented. It presents valuable information on the prevalence of community CPE in Pakistan. WGS highlights the dominant sequence types carrying CPE genes and provides epidemiological information on plasmids carrying carbapenemase genes.

I have the following comments and suggestions for the authors:

METHODS:

Page 5, line 98: Antimicrobial susceptibility testing. It would add to the manuscript if the authors could perform susceptibility testing against newer agents, like aztreonam+avibactam and cefiderocol.

Response: the association aztreonam+avibactam has been tested for a subset of CPE (NDM-5 producing E. coli), the results being reported in this article: "International circulation of aztreonam/avibactam-resistant NDM-5-producing Escherichia coli isolates: successful epidemic clones." Journal of global antimicrobial resistance 27 (2021): 326-328. Indeed, a significant number of strains combining NDM-5, CMY-42 and specific insertions motives in the PBP3 showed a decreased susceptibility to aztreonam-avibactam.

As for cefidérocol, it is not available in Pakistan. In France, we currently have a shortage for reagents required to perform microdilution tests (E-tests strips and discs being not recommended for céfidérocol testing). Nonetheless, we keep this in mind for our future work.

RESULTS:

Sampling: Page 8, line 160-162: In this study a young patient cohort was investigated (average age 38 and 44 years). How does this compare to other studies from the region? Could this influence the finding?

Response: We have added a paragraph regarding influence of young patient cohort on the findings in discussion part, also reference of few studies showing children and older people are at high risk of acquisition. We agree that the prevalence of CPE could be higher in at-risk populations such as elderly and young children. Page no. 15, L: 326-332.

Prevalence of CPE carriage: Page 8, line 169: It would be interesting to add some information on whether the high community prevalence was reflected in disease burden in the hospital. How many of the patients required treatment for CPE? Were any of the hospital acquired cases considered to be linked?

Response: Information has been added regarding situation of patients from which samples were collected on Page no. 9 (lines 181-182). Patients were admitted for or pregnancy-related planned consultation. We did not record the occurrence of infections during the follow-up, but given our results, such study would be interesting to implement. As for the connection between community and hospital-acquired strains, we only screened for hospital-acquired CPE in patients found negative at admission. To see whether community-acquired strains can cause hospital-onset infections, we should consider CPE-positive patients at admission. Of note, few community and hospital acquired cases were closely linked as mentioned on Page no. 11, lines 233-235.

Phylogeny: Page 10, line 220: The authors comment on the maximal genetic distances but do not discuss genetic similarities. On the tree some of the sequences look like they are related? Did they find possible relatedness between community and hospital acquired strains indicating possible cross transmission? Could the authors comment on this?

Response: This has been mentioned in the results section, Phylogeny at Page 11 (lines 234 – 237).

Relative abundance: Page 11, line 239: The authors present data on quantification of CRE but do not discuss the finding. Did they expect a difference in the relative abundance between community-acquired CRE and hospital-associated CRE? Does this depend on whether the patient receives antibiotics? I suggest to briefly mention this in the discussion.

Response: We have discussed these points in the discussion section on page No. 15-16 (lines 333-337).

DISCUSSION:

Line 248: ‘CPE carriage is mostly associated with hospital environment’ Did the other studies undertaken in Pakistan focus on hospital acquired cases? It would be interesting to discuss isolates that were acquired in hospital here. On the tree they look spread out over the clades but closely related to some of the community isolates? What is known about hospital acquisition of CPE in hospitals in Pakistan? How many of the CPE positive patients did require CPE treatment? What impact does the high community carriage rate have on hospitals?

Response: Limited data is available regarding community carriage of CPE in Pakistan and mostly studies are based on hospital isolates. Hospital acquired isolates have been mentioned in discussion, along with their relatedness with community associated isolates. Factors which are responsible for CPE acquisition in hospitals and impact high community carriage on hospitals have also been included in revised version. (please see Page No. 12-13, lines 260-269). Data about CPE positive patients requiring CPE treatment was not collected. The impact of high community carriage has been discussed on pages 12-13 (lines 260 – 269).

Line 278: ‘Combination of resistance genes, virulence, and ability to easily disseminate…’ The authors mention virulence here but do not discuss virulence data for the observed ST types. How frequent is ST 167 found in clinical samples?

Response: The word virulence has been removed from the description of ST 167. The prevalence and importance of ST 167 in clinical settings has been discussed in detail at page 14 (lines 298 – 300).

Line 295/296: It would add to the value of the manuscript to be able to discuss cefiderocol susceptibilities here. It might not be available as an agent in Pakistan yet but with the high prevalence of metallo-b-lactamases it would be helpful to assess its activity against the isolates of the study.

Response: Following the Reviewer 2’s comment on antibiotics potentially active on CPE, cefiderocol is not available in Pakistan as a drug or for testing. In France, we currently have a shortage for reagents required to perform microdilution tests (E-tests strips and discs being not recommended for céfidérocol testing). Nonetheless, we keep this in mind for our future work.

Besides, the association aztreonam+avibactam has been tested for a subset of CPE (NDM-5 producing E. coli), the results being reported in this article: "International circulation of aztreonam/avibactam-resistant NDM-5-producing Escherichia coli isolates: successful epidemic clones." Journal of global antimicrobial resistance 27 (2021): 326-328. Indeed, a significant number of strains combining NDM-5, CMY-42 and specific insertions motives in the PBP3 showed a decreased susceptibility to aztreonam-avibactam.

============================================================

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

7 Feb 2022

17 Feb 2022

Reviewer's comments:

Reviewer #1: There is incorrect on lines 327-328 "A study from Thailand also reported similar findings of mcr-1 detection from asymptomatic human feces in 2012 [52]." That study is conducted on patients with variety specimens, no feces. The author should be check and carefully mentioned. Please revise this sentence again.

Response: Thank you for pointing out this mistake. Correction has been made. Page 15, Line, 329.

-----------------------------------------------------------------------------------------

Reviewer #2:

Overall, it has improved, but there was one point that was not answered satisfactorily because the previous my review was unclear.

L. 150-158 Methods, Quantification of CRE

The counting method regarding the total amount of Gram-negative bacteria is unclear. According to the description in the text, rectal specimens were first inoculated onto ertapenem-supplemented agar plates. Therefore, even if the specimens were cultured on agar plates without ertapenem, the total amount of Gram-negative bacteria may not have been counted accurately, and those selected for ertapenem may have been counted. In such a case, the number of Gram-negative bacteria would be low and the CRE would be overestimated, which raises questions about the reliability of the CRE-RA and is unacceptable. The method of counting total Gram-negative bacteria should be clearly explained.

Response: Thank you for pointing out the confusion in methods. The rectal swabs were not directly plated on the plates. Instead they were thoroughly mixed in normal saline first and then 100 ul of the swab saline mix was plated on MacConkey agar with and without antibiotic directly and serial dilutions were also made to calculate relative abundance. This methodology was followed based on previously published methods in following articles: ‘Relative Fecal Abundance of Extended-Spectrum-β-Lactamase-Producing Escherichia coli Strains and Their Occurrence in Urinary Tract Infections in Women. Antimicrob. Agents Chemother. 57, 4512–4517 (2013)’ and ‘High Rate of Acquisition but Short Duration of Carriage of Multidrug-Resistant Enterobacteriaceae After Travel to the Tropics. Clin. Infect. Dis. 61, 593–600 (2015)’

The methods part has been re-phrased to make it more clear. Please see page No. 5, line 98 and page No. 7, lines 151-153.

============================================================

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

2 Mar 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-emailutm_source=authorlettersutm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Abdelazeem Mohamed Algammal, Prof, Ph.D

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #4: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #4: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #4: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #4: Comments to authors:

-The current study is interesting; however, the authors should address the following comments to improve the quality of the manuscript:

- Please write the scientific names of bacterial pathogens and genes in the correct form all over the manuscript and the references section.

Title:

I think the work would benefit from the title that contains the main conclusion of the study (should be derived from the conclusion). Please modify the title.

Abstract:

- The abstract must illustrate the used methods and the most prevalent results (give more hints about methods and results). Besides, rephrase the aim of the work and the main conclusion of your findings.

-Introduction: (it needs to be more informative):

-Give a hint about the virulence factors, different infections caused by E.coli and K. pneumoniae, and the mechanism of disease occurrence.

- The authors should illustrate the public health importance concerning the emergence of multidrug-resistant (MDR) bacterial pathogens that reflect the necessity of new potent and safe antimicrobial agents. Several studies proved the widespread MDR- bacterial pathogens;

Authors could add the following paragraph:

Multidrug resistance has been increased all over the world that is considered a public health threat. Several recent investigations reported the emergence of multidrug-resistant bacterial pathogens from different origins including humans, birds, cattle, and fish that increase the necessity of new potent and safe antimicrobial agents. Besides, the routine application of the antimicrobial susceptibility testing to detect the antibiotic of choice as well as the screening of the emerging MDR strains. You are advised to cite the following valuable studies:

1.PMID: 33177849

2.PMID: 33188216

3.PMID: 30150182

4.PMID: 33947875

5.PMID: 32994450

6. PMID: 32497922

7.PMID: 33061472

8.PMID: 34445951

9.https://doi.org/10.5114/ceji.2013.3774020 .

10.https://doi.org/10.1016/j.aquaculture.2021.737643

11.https://doi.org/10.1016/j.foodcont.2021.108066

-Rephrase the aim of the work to be clear and better sound.

Material and methods:

-Add this subtitle: Bacterial Isolation and identification:

•Discuss in detail the methods of isolation and identification of Enterobacterales, especially, E. coli and K. pneumoniae. Besides, specific references should be added.

•Add the company, city, and country of the used bacterial media and reagents that were used in the biochemical identification of isolates. Also, enumerate all used biochemical reactions.

- Modify the subtitile to be: Antimicrobial susceptibility testing:

-Illustrate the antimicrobial classes of the tested antibiotics.

-The authors are advised to classify the tested isolates to MDR , XDR, and PDR as described by Magiorakos et al.

Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012; 18:268–81. doi:10.1111/j.1469-0691.2011.03570.x.

- The correlation between phenotypic and genotypic multidrug resistance should be performed.

-Add the following subtitle to the methods section, and disscuss the method in detail ( Support the methods with specific references):

Multilocus sequence typing (MLST)

-Statistical analyses:

-Add more details about the software used in the statistical analyses.

-Results:

-Add this subtitle: Phenotypic characteristics of the recovered isolates:

•Illustrate in detail the phenotypic characteristics of the recovered E. coli and K. pneumoniae isolates.

- Illustrate in a table the results of The Antimicrobial susceptibility testing.

•Illustrate in a new table the occurrence of MDR (Multidrug resistance) among the recovered isolates as the following (illustrate the names of the antimicrobial classes and different antibiotics):

No. of strains%Type of resistance

R, MDR, and XDRPhenotypic multidrug resistance

(Antimicrobial classes and different antibiotics).The antibiotic -resistance genes

-The correlation (Correlation coefficient) between phenotypic and genotypic multidrug resistance should be performed.

- Add the supplemetary Figures to the main manuscript.

-Increase the resution of all Figures ( it should be 600 dpi).

-Discussion:

- The authors are advised to illustrate the real impact of their findings without repetition of results.

-Illustrate the different mechanisms of antimicrobial resistance in E. coli and K. pneumoniae.

-Conclusion

- Should be rephrased to be consise and sounded. A real conclusion should focus on the question or claim you articulated in your study, which resolution has been the main objective of your paper?

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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Reviewer #4: No

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15 Apr 2022

Reviewer #4: Comments to authors:

The current study is interesting; however, the authors should address the following comments to improve the quality of the manuscript:

Please write the scientific names of bacterial pathogens and genes in the correct form all over the manuscript and the references section.

Response: Bacterial pathogens and genes names have been re-checked and corrected where needed, Page No. 2, lines 43-44 and Page No. 21-27, lines 459, 462, 466, 472, 521, 529, 540, 583, 626, 630, 633, 643,646, 650, 653, 660, 664, 668, 672, 679, 683, 688.

Title:

I think the work would benefit from the title that contains the main conclusion of the study (should be derived from the conclusion). Please modify the title.

Response: This manuscript has been reviewed two times previously and we already made changes based on previous reviews. Furthermore, we found dissemination of many clones of bacteria, not just one specific; hence we are keeping a generalized title.

Abstract:

The abstract must illustrate the used methods and the most prevalent results (give more hints about methods and results). Besides, rephrase the aim of the work and the main conclusion of your findings.

Response: Methods have been added, Page No. 2, lines: 38-42 while most prevalent results have already been described in abstract. Please see Page No. 2, lines 43-49 and this was reviewed two times previously and we had changed it based on previous reviews. The main conclusion is re-phrased; Pages No. 2-3, lines: 49-53.

Introduction: (it needs to be more informative):

Give a hint about the virulence factors, different infections caused by E. coli and K. pneumoniae, and the mechanism of disease occurrence.

Response: Virulence factors, infections, and mechanism of disease occurrence have been added in introduction to make it more informative. Please see Page No. 3, lines 57-64.

The authors should illustrate the public health importance concerning the emergence of multidrug-resistant (MDR) bacterial pathogens that reflect the necessity of new potent and safe antimicrobial agents. Several studies proved the widespread MDR- bacterial pathogens;

Authors could add the following paragraph:

Multidrug resistance has been increased all over the world that is considered a public health threat. Several recent investigations reported the emergence of multidrug-resistant bacterial pathogens from different origins including humans, birds, cattle, and fish that increase the necessity of new potent and safe antimicrobial agents. Besides, the routine application of the antimicrobial susceptibility testing to detect the antibiotic of choice as well as the screening of the emerging MDR strains. You are advised to cite the following valuable studies:

1. PMID: 33177849

2. PMID: 33188216

3. PMID: 30150182

4. PMID: 33947875

5. PMID: 32994450

6. PMID: 32497922

7. PMID: 33061472

8. PMID: 34445951

9. https://doi.org/10.5114/ceji.2013.3774020 .

10. https://doi.org/10.1016/j.aquaculture.2021.737643

11 https://doi.org/10.1016/j.foodcont.2021.108066

Response: The suggested paragraph is a generalized information on AMR which is suitable for a study focusing on AMR and One health. Our manuscript is focused work investigating the carriage of CREs and CPEs in community and the related information on Enterbacterales and carbapenem resistance mechanisms have been described in the introduction. We have added information on virulence, and diseases as suggested in prior comments. We appreciated the advice to add valuable studies but the references advised above are not significantly relevant to our work and do not add any value to the work, hence these have not been added.

Rephrase the aim of the work to be clear and better sound.

Response: Aim of the work has been rephrased, Page No.4, lines 88-92.

Material and methods:

Add this subtitle: Bacterial Isolation and identification

Response: Subtitle and relevant detail has been added. Page No. 5, line no. 104.

Discuss in detail the methods of isolation and identification of Enterobacterales, especially, E. coli and K. pneumoniae. Besides, specific references should be added.

Response: The methods of isolation have already been discussed, Page No.5, line no. 112-115 while identification methods of Enterobacterales with references have been added, Page No. 6 line no. 115-126.

Add the company, city, and country of the used bacterial media and reagents that were used in the biochemical identification of isolates. Also, enumerate all used biochemical reactions.

Response: Biochemical reactions, company, city, and country of all used bacterial media and reagents have been added; Page No. 6, line no. 118-125.

Modify the subtitle to be: Antimicrobial susceptibility testing:

Illustrate the antimicrobial classes of the tested antibiotics

Response: Subtitle named antimicrobial susceptibility testing has been added, Page No. 6, line no. 128, and antimicrobial classes have been added to the material and method section under the heading of antimicrobial susceptibility testing; pages 6-7, line no. 130-141.

The authors are advised to classify the tested isolates to MDR, XDR, and PDR as described by Magiorakos et al.

Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012; 18:268–81. doi:10.1111/j.1469-0691.2011.03570. x.

Response:

Response: Categories of isolates have been made to XDR, and MDR as described by Magiorakos et al., Page No.12, lines 247-250. No isolate was found to be PDR.

The correlation between phenotypic and genotypic multidrug resistance should be performed

Response: The work aimed to assess the carriage of CREs and CPEs in community. Antibiotic susceptibility were analyzed for all isolates (n=170) while the detailed genomic analysis was done for 78 isolates after de-duplication based on antibiotic susceptibility profiles. The assessment of methodologies in detecting the resistance was not the scope of the work hence the correlation of phenotypic and genotypic methods has not been carried out in this work.

Add the following subtitle to the methods section and discuss the method in detail (Support the methods with specific references):

Multilocus sequence typing (MLST), Statistical analyses:

Add more details about the software used in the statistical analyses.

Response: Subtitles and relevant detail of MLST with references (Page No. 7, lines 155-158) and Statistical analyses along with references, and detail of software has been added; Page No. 9 line no. 192-197.

Results:

Add this subtitle: Phenotypic characteristics of the recovered isolates: Illustrate in detail the phenotypic characteristics of the recovered E. coli and K. pneumoniae isolates.

Response: Subtitle and characteristics of recovered Enterobacterales isolates have been added, Page No. 11, line no. 221-224.

.

Illustrate in a table the results of The Antimicrobial susceptibility testing

Table of antimicrobial susceptibility testing results has been added in supporting material as S1 Table (Page No. 12, line No. 243 and Page No. 28, line 690.

Illustrate in a new table the occurrence of MDR (Multidrug resistance) among the recovered isolates as the following (illustrate the names of the antimicrobial classes and different antibiotics):

No. of strains%, Type of resistance

R, MDR, and XDR Phenotypic multidrug resistance

(Antimicrobial classes and different antibiotics). The antibiotic -resistance genes

Response: Table showing Isolates categorization into XDR and MDR has been added in the supporting material (S2 Table: Page No. 28, line 691), showing number of strains, %, type of resistance and phenotypic multidrug resistance and susceptibility (Antimicrobial classes and different antibiotics), while detail of all antibiotic resistance genes has already been described in (S3 Table: Page No. 28, line no. 692).

The correlation (Correlation coefficient) between phenotypic and genotypic multidrug resistance should be performed.

Response: The work aimed to assess the carriage of CREs and CPEs in community. Antibiotic susceptibility were analyzed for all isolates (n=170) while the detailed genomic analysis was done for 78 isolates after de-duplication based on antibiotic susceptibility profiles. The assessment of methodologies in detecting the resistance was not the scope of the work hence the correlation of phenotypic and genotypic methods has not been carried out in this work.

Add the supplementary Figures to the main manuscript.

Response: All Supplementary figures have been added to main manuscript, Page No. 10, lines 215-217, Page No. 13, lines. 278-283, Page No. 15, lines.318-319, Page No. 16, line no. 326-328.

Increase the resolution of all Figures (it should be 600 dpi).

Response: Resolution of all figures has been increased to 600 dpi.

Discussion:

The authors are advised to illustrate the real impact of their findings without repetition of results.

Response: Impact of study findings has been added to discussion, Page No.20, Line no. 413-417.

Illustrate the different mechanisms of antimicrobial resistance in E. coli and K. pneumoniae.

Response: Prevalent resistance mechanisms in CRE have already been discussed in Introduction, Pages 3-4, line no. 71-80.

Conclusion

Should be rephrased to be concise and sounded. A real conclusion should focus on the question or claim you articulated in your study, which resolution has been the main objective of your paper?

Response: Conclusion has been improved according to suggestion, Pages 20-21, lines 427-435.

Submitted filename: Response to Reviewer.docx

Click here for additional data file.

13 May 2022

20 May 2022

RE: PLOS One (PONE-D-21-36109R3)

Title: Dissemination of Carbapenemase-producing Enterobacterales in the community of Rawalpindi, Pakistan

============================================================

Review Comments to the Author

Reviewer #1: This revised manuscript has been addressing all points of the reviewer's comments. I satisfy the response of the authors.

------------------------------------------------------------------------------------------------------------------------------------

Reviewer #2: My concerns previously noted have been corrected, and together with the corrections indicated by Reviewer 4, the manuscript is much better. I have commented on the parts of the text that the author refused in response to Reviewer 4's remarks as follows.

The comment refused by authors: Title

Comments: No modification is required. I do not believe that the title change is necessary because, as the authors claim, the study investigated the genus Enterobacterales bacteria.

The comment refused by authors: References assigned by Reviewer 4

Comments: No modification is required. The references assigned by Reviewer 4 are biased in authorship and are not impartial. I believe they should NOT be added.

The comment refused by authors: Discuss in detail the methods of isolation and identification of Enterobacterales, especially, E. coli and K. pneumoniae.

Comments: Modification is required. Selection plates and concentration of antimicrobials used will affect the detection rate. It is a good idea to add this information to the “limitation” in discussion.

Response: The methods of isolation and identification of Enterobacterales have already been discussed page No. 5-6, lines 102-125. The limitation of selection plates and concentration of antibiotics has been added to the limitation of study on Pages 19-20. lines 415-418.

The comment refused by authors: The correlation between phenotypic and genotypic multidrug resistance should be performed

Comments: Modification is required. I think, Reviewer 4 may not have pointed out the methodologies. The paper does include antimicrobial susceptibility tests and a genetic study, which suggests that the necessary experiments have been performed. However, it does not present data linking the phenotypic antimicrobial susceptibility to the genotype. In particular, a number of strains have been found to be susceptible to monobactam, aminoglycosides, and imipenem, and the correlation with genotype needs to be discussed.

Response: The data of the observed phenotypic resistance along with the genomic content of the antibiotic resistance genes has been added. Since this information is not in the scope of main findings of the study, this has been included in the supplementary information as S4 Table.

The comment refused by authors: Illustrate the different mechanisms of antimicrobial resistance in E. coli and K. pneumoniae.

Comments: No modification is required. As the author claims, the drug resistance mechanism is introduced in the introduction, so there is no need to add it to the discussion again.

----------------------------------------------------------------------------------------------------------------------------------

Reviewer #3: (No Response)

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

16 Jun 2022

Dissemination of Carbapenemase-producing Enterobacterales in the community of Rawalpindi, Pakistan

PONE-D-21-36109R4

Dear Dr. Zahra,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Jianhong Zhou

Staff Editor

PLOS ONE

Additional Staff Editor Comments: Please  update your data availability statement on the submission details page by including the accession number PRJNA645311 as this will be used for the Data Availability Statement in the published article.   statementonthesubmissiondetailspageasthiswillbeusedfortheDASinthepublishedarticle.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Masahiro Suzuki

**********

30 Jun 2022

PONE-D-21-36109R4

Dissemination of Carbapenemase-producing Enterobacterales in the community of Rawalpindi, Pakistan

Dear Dr. Zahra:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

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on behalf of

Jianhong Zhou

Staff Editor

PLOS ONE