Household cockroaches carry CTX-M-15, OXA-48 and NDM-1, and share beta-lactam resistance determinants with humans

Aim Household insect pests, including cockroaches, have gained consideration as potential vectors for multidrug resistant pathogens of public health concern. This study was designed to investigate whether household cockroaches share beta-lactam resistance determinants with human inhabitants. Methods From February through July 2016, 400 cockroaches were systematically collected from 100 households. Whole insect homogenates and faecal samples from inhabitants of all included households were cultured for cephalosporin-resistant enterobacteria (CRe). The CRe were examined for AmpC, ESBL, and carbapenemase genes; antibiotic susceptibility patterns; and conjugative transfer of antibiotic resistance mechanisms. Clonal relationships between isolates were determined by multi-locus sequence typing (MLST). Results Twenty CRe were recovered from whole cockroach homogenates of 15 households. Five harbored ESBL genes (2 blaCTX-M-15/TEM-1; 1 blaCTX-M-15/TEM-4; 1 blaTEM-24; 1 blaSHV-4), and 3 carried carbapenemase genes (2 blaNDM-1 genes and 1 blaOXA-48 gene) all of which were transferrable by conjugation to E. coli J53 recipients. There was high clonal diversity with low inter-species similarity regardless of the beta-lactamase gene sequence. From 6 households, the pair of cockroach and human CRe shared the same antibiogram, ST and/or conjugable blaESBL gene sequence (house 34, E. coli ST9-blaTEM-4; house 37, E. coli ST44-blaCTX-15/TEM-4; house 41, E. coli ST443-blaCTX-15/TEM-1; house 49, K. pneumoniae ST231-blaSHV-13). Conclusion The findings highlight household cockroaches as reservoirs of CTX-M-15, OXA-48 and NDM-1 genes that share beta-lactam resistance determinants with humans.


Introduction
The production of extended-spectrum beta-lactamases (ESBL), Class C cephalosporinases and 3 carbapenemases constitute the primary antibiotic resistance mechanism in Enterobacteriaceae 4 [1,2]. Together, these beta-lactamases confer resistance to all available beta-lactam antibiotics, and 5 are associated with significantly high levels of co-resistance to other classes of broad spectrum 6 antimicrobials [1-6]. Increasingly, the CTX-M-15 type ESBLs are becoming predominant in 7 Ghana [7,8]. Occurrence of the recently described OXA-48 type carbapenemase and widespread 8 reports of bla NDM-1 genes across Africa further compound the outlook of the antibiotic resistance 9 problem [9][10][11][12][13]. Indeed, emergence of such resistant bacteria is often related to misuse and abuse 10 of beta-lactam antibiotics [14,15]. However, the burden of such pathogens is also hugely aided by 11 poor sanitation [16][17][18]. Cockroaches, which widely colonize the environment, including human 12 dwellings, may well act as vectors of antibiotic-resistant bacteria. 13 Cockroaches are abundant in Ghanaian households and are known to harbor an array of pathogens 14 [19]. They often reside in household sewage pipe systems, which are a repository of diverse 15 infectious microorganisms. That cockroaches carry pathogenic bacteria is well documented in 16 literature [19][20][21][22][23]. Some of the pathogenic bacteria may be carriers of drug-resistance 17 determinants, particularly beta-lactamases. However, only few studies have investigated AmpC, 18 ESBL or carbapenemase resistance elements in bacteria from cockroaches [24]. Consequently, the 19 vector potential of cockroaches in the dissemination of such multidrug-resistance mechanisms is 20 largely under-reported. In this study, we report the presence of CTX-M-15-, OXA-48-and NDM-  Sample collection and processing 17 All 100 households were provided with 100 ml sterile containers with screw caps, sterile zipper 18 bags containing gloves and forceps to use for capturing cockroaches. A member of each household 19 was selected and educated appropriately to be responsible for capturing cockroaches. Only live 20 cockroaches found indoors were captured for this study. Four cockroaches (irrespective of species) 21 were requested per household and pooled as one sample. Each cockroach sample was soaked in with a sterile rod. The triturate was once again vortexed vigorously for 6 minutes to obtain a whole 2 insect homogenate. A loop-full (approximately 10 µL) of suspension from each sample was 3 inoculated onto SSI agar plate (SSI, Diagnostica, Denmark) with 30 µg cefpodoxime disk 4 (MAST, UK) and incubated overnight at 37 o C. For stool samples, 1 gram of each specimen was 5 suspended in 10 ml of sterile 0.9% physiological saline and vigorously vortexed. 1 ml of the 6 suspension was cultured on SSI agar plates with 30 µg cefpodoxime disk. From each culture plate, 7 distinct morphological phenotypes of enterobacteria growing around the cefpodoxime disks 8 within an inhibition zone of 21mm were defined as screen positive for third generation 9 cephalosporin resistance (CRe). Each distinct morphological phenotype was identified to the 10 species level using the biochemical test kits Minibact-E® (SSI, Diagnostica, Denmark) according 11 to the manufacturer's guidelines. Subsequently, four colonies of each speciated phenotype were 12 subjected to the Kirby-Bauer method of susceptibility testing per guidelines of the Clinical and 13 Laboratory Standard Institute (CLSI) using cefotaxime (30 µg) and ceftazidime (30 µg) 14 antibiotics [27]. Isolates resistant to cefotaxime or ceftazidime were confirmed as CRe.

15
Susceptibility test and assays for ESBL, AmpC and carbapenemase 16 All CRe isolates were tested for susceptibility to the following antibiotics (MAST, UK) according

18
All CRe isolates were included in the conjugation assay. Conjugations were done using sodium 19 azide-resistant E. coli J53 as recipient [34]. None of the CRe showed resistance to sodium azide.

20
The donors were cultured in Luria-Bertani (LB) (MAST, UK) broth with cefotaxime (8 µg/ml) 21 overnight. The recipient was also cultured overnight in LB broth but with no antibiotic.

22
Subsequently, 1 mL aliquots of each donor and the recipient were separately transferred into fresh with an equal volume of the recipient and the mixture was incubated for 6 hours at 37 o C. Selection 2 for transconjugants was carried out on MacConkey agar supplemented with sodium azide (150 3 mg/L) and cefotaxime (8 mg/L) or cefoxitin (32 mg/L) or meropenem (2 mg/L). Transconjugants 4 were confirmed for ESBL, AmpC or carbapenemase genotype as previously described.

21
Of the 20 cockroach CRe, 12 isolates (from 10 samples) expressed ESBL (n=5), AmpC (n=4) or 22 were resistant to meropenem (n=3) ( Table 1). The AmpC-producers were Enterobacter freundii 23 (n=2) and Enterobacter agglomerans (n=2). Isolates with ESBL phenotype included 4 E. coli and 1 1 K. pneumoniae. Two of the 3 meropenem resistant isolates (E. freundii and C. cloacae) were 2 found to be metallo-beta-lactamse (MBL)-producers. The third (E. coli) was non-susceptible to 3 temocillin (30 ug); and was deemed presumptively positive for OXA-48 like carbapenemase 4 ( Table 1). None of the CRe isolates was positive for any combination of the three phenotypes. Of  (Table 1). The CRe without any of the phenotypes were 12 MDR phenotypes in 3 of 8 isolates. They were susceptible to most of the non-beta-lactam 13 antibiotics, with all 8 isolates susceptible to gentamicin (n=7/7), tigecycline (n=7/7), 14 chloramphenicol (n=8/8), and nitrofurantoin (n=7/7). In contrast, MDR phenotype was reported in 15 all isolates with ESBL or AmpC phenotype. Two of the 3 carbapenemase-positive CRe were also 16 XDR. Table 2 shows the molecular characterization and antibiogram of all 20 human CRe. None    diversity with low inter-species similarity regardless of the beta-lactamase gene sequence (Fig 2).   were susceptible to several antibiotics tested. None of these CRe also successfully transferred their 4 cephalosporin resistance phenotype to E. coli J53 recipients by conjugation, suggesting an intrinsic  Our data should be interpreted considering potential limitations. Households were provided with 10 cockroach collection kits and procedural guidelines. We are however unable to ascertain if 11 participants strictly adhered to aseptic collection guidelines. Nonetheless, the fact that all the CRe 12 isolates were the dominant colonies on culture suggest stable colonization. We have no data on 13 antibiotic use by household contacts and are thus unable to relate CRe colonization and antibiotic 14 consumption. Due to small sample size and provincial household concentration, our study is not 15 meant to be representative for the prevalence of AmpC-, ESBL-, or carbapenemase-producing 16 bacteria. It is noteworthy that the complimentary sampling of humans is biased since only humans 17 with corresponding positive household cockroach samples were examined. The study was 18 designed to investigate whether, within individual households, cockroaches and humans share 19 isolates of the same clone and antibiotic resistance determinants of public health concern 20 Conclusion. We report the disturbing colonization of household cockroaches with multidrug public health concern; and calls for regulations on their control especially in healthcare settings.